Project Summary/Abstract The enteric nervous system (ENS) is a complex network of peripheral neurons and glial cells that controls gut motility by coordinating the involuntary contraction and relaxation of innervated smooth muscle along the length of the gastrointestinal tract. While the ENS of the intestine is particularly well-studied, the development, function, and disease pathologies of more proximal ENS are largely unstudied. The preganglionic control of the esophagus has been studied at the level of the CNS and the vagus nerve that controls distal GI motility, but the peripheral neuroglial network that is embedded within the foregut, the relative distribution neuronal subtypes and glia, and the basic architecture of how the ENS interfaces with the postsynaptic tissues in the esophagus and stomach are largely undetermined. Moreover, the basic process by which the enteric neuroglial network of the proximal ENS is assembled during embryonic development is virtually unstudied. Understanding how neurons connect with its target cell types will provide critical insight into how the proximal ENS functions postnatally. Additionally, developmental studies were foundational for recent successes in engineering human small intestinal tissues with a functional ENS. While these types of approaches to study innervation of the foregut have not been done, they are critical if we are to understand peripheral control of foregut tissue function. In this proposal, we aim to 1) map the molecular and cellular architecture of the proximal ENS, 2) identify mesenchymal signals that control proximal ENS development, and 3) generate human foregut tissues with a functional ENS. We have identified that mouse embryos lacking the transcription factor Osr1 lack an ENS in the proximal gut. While esophageal and gastric epithelium and mesenchyme are present in these e13.5 Osr-/- embryos, enteric neurons and smooth muscle are virtually absent. In our first aim, we will identify the primary developmental defects of proximal ENS development in Osr1 mutant embryos. Based on our transcriptional analysis of foregut tissue isolated from e9.5 Osr-/- embryos, we hypothesize that Osr1 functions predominantly in the mesenchyme to control ENS development. We will use a mouse genetic approach to delete Osr1 specifically in mesenchyme, as well as in ENCCs and endoderm, to identify germ-layer specific roles of Osr1 in proximal ENS development. We have also identified several signaling pathways, which are known to regulate ENS formation, that are perturbed in e9.5 Osr-/- embryos, including RA, Semaphorin, and BMP. We hypothesize that Osr1 mutant mesenchyme has disrupted expression of key signaling molecules that act in a paracrine fashion to control proximal ENS development. In our second aim, we will use a highly manipulable in vitro culture system, to examine the effects of Osr1-regulated signaling pathways on proximal ENS development. We will generate human esophageal organoids with a functional ENS to model normal and perturbed assembly of proximal ENS. We will manipulate these pathways in ENCC cultures recombined with esophageal organoids and examine the resulting perturbations in ENS development.